Cholesterol absorption was measured in 94 normal subjects aged 17-80 while consuming diets low in cholesterol (mean intake=226(126 mg/day). A new dual stable isotope method was used in which 15 mg cholesterol tracer with 6 additional mass units ([26,26,26,27,27,27--2H6]cholesterol) was given intravenously and 30 mg of another tracer with 5 additional mass units ([2,2,4,4,6-2H5]cholesterol or [23,24,25,26,27-13C5]cholesterol) was given orally during a test meal. The ratio of tracers in plasma was determined by negative ion mass spectrometry of pentafluorobenzoyl sterol esters. Absorption values ranged from 29.0% to 80.1% (mean 56.2(12.1). Cholesterol absorption was increased in African-Americans (63.4(11.8% vs. 55.1(11.9%, p=0.027) but was similar for women (53.3(11.9%) and men (57.6(12.1%). It was not related to plasma lipoproteins, age, apo-E genotype, or chronic dietary intake of energy, fat, or cholesterol quantitated from food records. The amount of dietar y choleste rol absorbed was positively correlated with fasting plasma insulin (r=0.525, p<0.0001), C-peptide (r=0.367, p=0.0003) and glucagon (r=0.421, p<0.0001), independent of gender, body fat percent and age. Efficiency of intestinal cholesterol absorption and amount of dietary cholesterol absorbed were not related to plasma or LDL cholesterol in individuals consuming a low-cholesterol diet. The dominant factor determining dietary cholesterol absorption was intake rather than absorption efficiency.